CB1 cannabinoid receptors mediate the psychoactivity of cannabis, the most commonly abused illicit drug, and are also attractive therapeutic targets. Sustained CB1 receptor activation, as happens with heavy cannabis use, or if CB1 agonists are used therapeutically on a regular basis, leads to the development of behavioral tolerance - that is, more drug is required to produce the same effect. Earlier studies by us and others in simple preparations (e.g., cell culture models) indentified two serines in the C-terminus of the CB1 receptor whose phosphorylation desensitized CB1 signaling. To test the hypothesis that phosphorylation of these residues was necessary for the development of behavioral tolerance in an animal, we made a knockin mouse mutating these two residues to alanine (S426A/S430A). In preliminary experiments we have found that these mice, compared to wildtype mice are more sensitive to ?9tetrahydrocannabinol (THC), develop tolerance to repeated administration of THC more slowly and also recover from tolerance more rapidly. In the proposed work we will use these mice as a novel model system to address significant questions relating to the development and expression of tolerance to CB1 receptor agonists by completing three specific aims: 1. What is the role of CB1 receptor serines 426 and 430 during the development of tolerance to cannabinoids? By using our S426A/S430A knockin mouse we will determine the role of phosphorylation of these two serines in the acute response to cannabinoids, in the development of cannabinoid tolerance and dependence, and in the severity of precipitated withdrawal. 2. How does preventing phosphorylation of serines 426 and 430 affect CB1 signaling during tolerance? In these experiments we will use biochemical and electrophysiological approaches (GTP?S autoradiography, MAP kinase activation, and whole cell patch clamp of cultured neurons and in slices) to assess the biochemical signature of CB1 tolerance and its modification in the S426A/S430A mouse. 3. What is the mechanism of delayed tolerance? In our preliminary studies we found that while tolerance was slowed in the S426A/S430A knockin mice, it still developed. This delayed tolerance is likely to be important in the adaptive responses to chronic CB1 receptor stimulation. In this aim we will take a discovery approach to identify potential mechanisms underlying delayed tolerance. We will use microarrays to determine the transcriptional changes that take place as tolerance to THC develops in wildtype and S426A/S430A knockin mice. We will also use mass spectrometry to test the hypothesis that delayed tolerance is due to additional CB1 receptor phosphorylation. Tolerance in the clinical setting is significant and often difficult to manage problem. The completion of these studies will be helpful both for managing tolerance to CB1 agonists as it occurs in the therapeutic setting as well as for understanding the consequences of sustained and high intensity recreational cannabis use.